Cell culture is an in-vitro technique of cultivating cells under controlled conditions and in sterile conditions. The technique was first developed by Ross Harrison in 1907. During the process of cell culture, the cells are detached from their tissue of origin and segregated from each other, either by mechanical or enzymatic means. The detached cells are then transferred before culturing them in cell culture plate or flask. The first generation of producing cell through this method is called primary cell culture. The cells from the primary culture are transferred to a new culture plate with a fresh medium to enable room for more growth. After the first subculture, the primary culture becomes cell line. When a subpopulation of cells from the cellline is selected for expansion through growth in the cell culture medium, the culture of cells so obtained have more or less similar genotype and phenotype. This gives rise to cell strains. ("Cell Culture & Its Application")
Very often, cell strains have a genotype that help them undergo finite cell division. Cell line prepared from cancer cells, give rise to cell strain in which the cells are immortal and undergo multiple number of cell division. The cells in a culture can be divided on the basis of their morphology into three basic type: fibroblastic cells, epithelial like cells and lymphoblast like cells. Fibroblast and epithelial cells grow attached to the surface. The fibroblast cells have an elongated shape, and epithelial cells have a polygonal shape. Lymphoblast cells have a spherical shape and grow as cell suspension, without being attached to the surface. ("Cell Culture & Its Application")
Cell culture procedure:
Certain basic conditions have to be fulfilled in order to culture cells in-vitro. The first is the growth medium and the other are the incubation temperature and conditions. The basal growth medium has the optimum level of ions and amino acids that are required for growth. Glucose and glutamine enrichment in the growth media can promote cell survival. The optimum level of glucose for cell growth is 1-4mM glucose and 2mM glutamine respectively. The ideal pH of the medium for growth of cells is between 7.2 to 7.4. The other important condition for cell culture are the incubation temperature, CO2 and O2 supplied to culture vessels. ("Cell Culture & Its Application")
Cell culture technicians are trained in aseptic technique. This helps to prevent contamination of the cells and also ensure protection of the operator from infectious materials in the cell culture. Contamination reduces cell growth and promotes cell death. Contaminated cultures are not suitable for use in biological applications. All glassware and media used in the cell culture must be sterile. Certain cell culture handling practices like avoiding spill and splashes, while pouring or transferring media are also practiced to avoid contamination. ("Cell Culture & Its Application")
96-well cell culture plates are used for serial dilution and culture of cells. Serial dilution using 96 well plate is more suitable for cells that are not dependent on attachment. Serial dilution of cells using this technique is fast and easy. In some cases, 96 well cell culture plates are also used for attached cell culture. But the procedure is less efficient. The plates are also used in research, where the experiment is conducted on known number of cells in a culture well. 96 well plates are also suitable for use in drug testing and toxicity studies. ("Cell Culture & Its Application")
Application of cell culture:
Cell culture is widely used in molecular biology and cellular biology application like metabolic and physiological studies, testing of drugs, toxicity studies, mutagenesis and carcinogenesis. In industries, it is used for large scale screening of drugs, for preparation of vaccine, proteins, and other biological products. (Uppangala)
Cell culture is an important part of cancer research. Normal cells are converted into cancer cells using radiation, chemicals and viruses. These cells are then used to study cancer characteristics. Knowledge from in-vitro cell culture studies are translated to clinical solutions. ("Cell Culture & Its Application")
Results:
Calculation and result:
27 x 104 = 54.000 cells/ ml
= 54.000 cells/ 1000 microliter
1 cell = 0.0185 microliter
50.000 cells = 925 microliter
12.500 cells = 185 microliter
Minus Resazurin control from all sample:
Use these values to create a graph
Discussion:
What is the function of the class 2 biosafety cabinet?
The downward flow of HEPA filtered air, is effective in preventing cross contamination. The air that passes the HEPA filter is particulate free and is recirculated through the cabinet or is discharged via a canopy connection to the outside of the building. Usually a small portion of the air is recirculated, while the remaining air is discharged to the exterior. In certain circumstances, the air is sterilised before discharge to the external environment. (NUAIR)
2. What is the purpose of the cell subculture procedure?
Cells in the culture are maintained regularly through subculture. This is done to prevent cell death. Overgrowth can accelerate cell death in the culture. Cellines grow as a sheet of monolayer. Cell in the cellline are attached to the neighbouring cells and the cell culture surface. Regular sub culturing of cells, prevent overgrowth and accelerated cell death. The medium is also exhausted when there is overgrowth. Subculture is done when the adherent cells have covered the available cell surface area. During subculture, adherent cells are detached from the cell culture surface. Trypsin and mild mechanical force is used for detachment of the cells. The extent of cell to cell and surface adherence, will vary with the type of cell culture. The cells are very sensitive to enzymatic and mechanical damage. The time of contact with trypsin is optimised to prevent damage to the cells. Over exposure to trypsin can harm the cells. ("Types of Subculture of Cell: 2 Types")
3. Resazurin flurometric cell viability assay is a simple, sensitive, and rapid way of estimating cell viability. The non-fluorescent dye resazurin is converted into a red fluorescent product resorufin, by the viable cells in the culture medium. When there is no cell growth, the dye remains in its oxidised state, which is colourless, thus giving a negative resazurin reaction. The negative control in this case is the cell culture medium without the cells. The positive control is the cell culture medium, with a known amount of viable cells. It is important to decide on the optimum dilution of cells that can be used in positive control. Too many viable cells can further reduce resorufin to hydro-resorufin, which is again non-flurometric. When there is continuous growth in the medium, the rezarufin is reduced and the rate of reduction is proportional to the cell undergoing growth. The reduced dye is monitored by measuring the OD at 600nm and 570nm. The background OD at 600nm is subtracted from the OD at 570nm. The intensity of signal is proportional to the number of viable cells in the sample. (Borra, Ricardo Carneiro et al. 255-262)
What effect does the absence of NBCS have on the Balb cells?
New born calf serum provides the necessary nutrient and growth factors required for the growth of Balb cells. It promotes healthy growth in the cells. The serum also inactivates trypsin activity and prevents damage caused to cells by trypsin. ("Use of Fetal Calf Serum")
Inference: In the given cell culture experiment, we got 27 cell per well, and this is low concentration.
Conclusion: Cell culture is the process of culturing cells in-vitro. This technology has been instrumental in the development of molecular biology and cell biology disciplines. Adherence to certain cell culture practices like asepsis, growth medium requirements, incubation temperature, CO2 and O2 requirement, are very important for the culture of cells. Class 2 biosafety cabinets are used for cell culture purpose. They offer protection to the professional, biological product and the environment.
Work Cited
Borra, Ricardo Carneiro et al. "A Simple Method To Measure Cell Viability In Proliferation And Cytotoxicity Assays". Brazilian Oral Research, vol 23, no. 3, 2009, pp. 255-262. Fapunifesp (Scielo), doi:10.1590/s1806-83242009000300006.
"Cell Culture & Its Application". Aquafind.Com, 2017, http://aquafind.com/articles/Cell_Culture.php.
"Introduction To Biological Safety Cabinets". 2002, https://www.bakerco.com/introduction- biological-safety-cabinets.
NUAIR,. "How Class II Biological Safety Cabinets Work | Nuaire". Nuaire.Com, 2017, https://www.nuaire.com/how-class-two-bsc-work.
"Types Of Subculture Of Cell: 2 Types ". Biology Discussion, 2017, http://www.biologydiscussion.com/cell/types-of-subculture-of-cell-2-types-with- diagram/10514.
Uppangala, Nidhi. "Applications Of Animal Cell Culture Technique". Biotecharticles.Com, 2017, http://www.biotecharticles.com/Others-Article/Applications-of-Animal-Cell-Culture- Technique-366.html.
"Use Of Fetal Calf Serum". Humaneresearch.Org.Au, 2017, http://www.humaneresearch.org.au/campaigns/fetal_calf_serum.
